The invention relates to the use of synthetic immune response modifiers to induce the maturation of dendritic cells in vitro. The invention additionally relates to methods of maturing dendritic cells, to methods of enhancing the antigen presenting ability of dendritic cells, and of enhancing T-cell stimulation using synthetic immune response modifiers. The invention further relates to cellular adjuvants prepared with the dendritic cells that have been matured according to the method of the invention.
Dendritic cells are known to play an important role in the immune system, both for their potent antigen presenting ability and their ability to initiate T-cell mediated immune responses. Indeed, dendritic cells (xe2x80x9cDCxe2x80x9d) activate T-cells more efficiently than any other known antigen presenting cell, and may be required for the initial activation of naxc3xafve T-cells in vitro and in vivo. These cells are generally present in the body at locations that are routinely exposed to foreign antigens, such as the skin, lung, gut, blood, and lymphoid tissues. In general, DC are broadly classified as immature or mature. Immature DC endocytose and process antigen efficiently, but express low levels of costimulatory molecules. In contrast, mature DC display increased levels of costimulatory molecules CD40, CD80 and CD86, as well as HLA-DR. In addition, mature DC express CD83 and secrete increased amounts of various cytokines and chemokines that aid T-cell activation.
In addition to naive T-cell activation, DC can influence the balance of the Th1/Th2 immune response. Several reports have indicated that DC preferentially activate Th1 responses, with the major determining factor being IL-12 secretion from the activated DC. Macatonia et al., J. Immunol. 154:5071 (1995). Hilkens et al., Blood 90:1920 (1997). Other reports have shown that DC can induce the generation of either Th1 or Th2 clones. Roth, et al., Scand. J. Immunol. 43:646 (1996). The evidence indicates that multiple factors influence the ability of DC to initiate a Th1 or Th2 response, including the DC to T-cell ratio, the DC tissue of origin, the amount of antigen used to prime the DC, the expression of costimulatory molecules and the antigen injection route.
The pivotal role played by DC in antigen presentation and T-cell activation has resulted in considerable interest in the use of DC in immunotherapy. This is particularly evident in the areas of vaccinology and cancer immunotherapy. Although much effort has been devoted to the development of successful vaccines using recombinant DNA, successful clinical use of DNA vaccines has not been achieved. Recent evidence indicates that effective immunization with DNA vaccines requires recombinant protein expression from DC. Further, enhanced immunity in animal models has been achieved utilizing DNA vaccines that encode for cytokines or that contain CpG oligonucleotide sequences that upregulate DC maturation. Recently, autologous DC obtained from cancer patients have been used for cancer immunotherapy. See, e.g., WO98/23728. Accordingly, efficient ex vivo methods for generating DC are prerequisite for successful immunotherapy.
In general, the process of ex vivo DC generation consists of obtaining DC precursor cells and then differentiating the cells in vitro into DC before introduction back into the patient. However, the DC must be terminally differentiated, or they will de-differentiate into monocytes/macrophages and lose much of their immunopotentiating ability. Ex vivo DC maturation has been successfully accomplished with monocyte conditioned medium; recombinant cytokines such as TNF-xcex1, IL-1 and IL-6; bacterial products such as LPS, bacterial DNA and cross-linking CD40; and transfection with genes that encode cytokines or costimulatory molecules. While these methods are capable of producing mature DC, there are disadvantages to using recombinant molecules and cellular supernatants for maturing DC. These include inconsistent quality and yield from lot to lot of these reagents and the introduction of exogenous proteins into patients, which may be toxic or result in autoimmunity. Such reagents can also be expensive to produce, making the cost of immunotherapy prohibitively expensive. There is a need for a method of maturing DC in vitro that is reliable and efficient, without the drawbacks of the currently known methods.
We have found that certain immune response modifier (IRM) compounds can induce the maturation of DC in vitro. These compounds are small molecules that can be readily produced at a consistent, high level of purity and potency. By using these compounds one can efficiently and consistently mature DC, which can then be used as immunotherapeutic agents. The IRM compounds useful in the method of the invention are generally of the imidazoquinoline type; that is, they have a structure that contains the imidazoquinoline ring system or a similar ring system, such as imidazopyridine or imidazonaphthyridine.
Accordingly, the invention provides a method of in vitro maturation of dendritic cells comprising treating said dendritic cells with an imidazoquinoline type immune response modifying compound, as well as a population of dendritic cells produced by this method.
The invention further provides a method of enhancing the antigen presenting ability of dendritic cells comprising treating said dendritic cells with an imidazoquinoline type immune response modifying compound.
In addition, the invention provides a method of preparing a cellular adjuvant for the treatment of a disease comprising the steps of maturing dendritic cells in vitro by treating the dendritic cells with an imidazoquinoline type immune response modifying compound and exposing the mature dendritic cells to an antigen associated with said disease.